The primary transcripts of eukaryotic structural genes (precursor mRNAs; pre-mRNAs) contain intervening sequences (introns) that are removed by RNA splicing. In some instances, alternative splicing of a common pre-mRNA provides an important mechanism to regulate gene expression. We are particularly interested in the early steps of spliceosome assembly, which play key roles in selection of sp1ice sites and in splicing regulation. The U2 snRNP Auxiliary Factor (U2AF) is an essential splicing factor that binds to the polypyrimidine (Py) tract/3' splice site and initiates spliceosome assembly. Experiments are proposed to further our understanding of U2AF RNA recognition, to characterize U2AF associated proteins, and to analyze how U2AF promote spliceosome assembly and splicing. During the past budget period, our studies on U2AF led to the cloning of novel human protein, hUAP56, a member of the DExD/H box family of RNA-dependent ATPases, and subsequently to the identification of a hUAP56 homologue in yeast, yUAP/Sub2p. We will use biochemical and genetic approaches to study human hUAP56 and yeast yUAP/Sub2p. By searching the recently available human genome sequence, we have identified four novel genes that encode proteins highly related to the small subunit of U2AF (U2AF35) and a related gene for UAP56. Experiments are proposed to study the expression and function of these recently discovered proteins. Splicing enhancers are cis-acting elements involved in constitutive and regulated splicing. They function by providing binding site for the SR family of proteins that contain arginine-serine rich (RS) domains. Experiments are proposed to study how RS domains promote spliceosome assembly and splicing.